1. Field of the Invention
The present invention relates to systems and methods for minimizing and/or eliminating diffusion effects in a microfluidic system. More specifically, embodiments of the present invention relate to systems and methods for minimizing and/or eliminating diffusion effects in a microfluidic system having one or more channels so that concentration dependent measurements can be made on a segmented flow of multiple miscible fluids in the one or more channels.
2. Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase Chain Reaction (“PCR”) is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies of the amplified DNA to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Once there are a sufficient number of copies of the original DNA molecule, the DNA can be characterized. One method of characterizing the DNA is to examine the DNA's dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA). The process of causing DNA to transition from dsDNA to ssDNA with increasing temperature is sometimes referred to as a “high-resolution temperature (thermal) melt (HRTm)” process, or simply a “high-resolution melt” process. Alternatively, the transition from ssDNA to dsDNA may be observed through various electrochemical methods, which generate a dynamic current as the potential across the system is changed.
Microfluidic chips are being developed for “lab-on-a-chip” devices to perform in-vitro diagnostic testing. The largest growth area is in molecular biology where DNA amplification is performed in the sealed channels of the chip. Optical detection devices are commonly used to measure the increasing amplicon product over time (Real Time PCR) and/or to perform a thermal melt to identify the presence of a specific genotype (High Resolution Thermal Melt). Exemplary disclosures related to the imaging of a microfluidic chip to measure the fluorescent product can be found in commonly-owned U.S. application Ser. No. 11/505,358 to Hasson et al. entitled “Real-Time PCR in Micro Channels” (U.S. Pat. Pub. 2008-0003588) and U.S. application Ser. No. 11/606,204 to Hasson et al. entitled “Systems and Methods for Monitoring the Amplification and Dissociation Behavior of DNA Molecules” (U.S. Pat. Pub. 2008-0003594), the respective disclosures of which are hereby incorporated by reference.
When a fluid is introduced into a channel to measure increasing amplicon product in the fluid over time and/or to identify the presence of a specific genotype in the fluid, it is desirable to minimize and/or prevent contamination of the fluid so that accurate results may be obtained. At the same time, it may be desirable to introduce a series of fluid species into a channel so that a single channel may be used to measure and/or identify multiple fluid species in succession. Minimization and/or prevention of contamination becomes especially difficult when the multiple fluid species are miscible (i.e., capable of being mixed) and are supplied to a single channel in a segmented fashion (i.e., with each species occupying the entire width of the channel and existing axially down or upstream from another of the species, which also occupies the entire width of the channel).
Flow through a microfluidic channel is generally characterized by laminar flow with parabolic velocity profiles. These parabolic velocity profiles indicate that fluid along the walls of the microfluidic channel will move much slower than the fluid at the center of the channel. In flows of only one species of fluid where the same chemical concentration exists at all points in the flow, this variation in fluid velocity as a function of distance from the channel wall has little impact. In flows in which two miscible species of fluid exist in a segmented fashion, the effects of the laminar velocity profile are problematic. In particular, as the fluid in the center of the channel moves faster than the edges, each segment of flow will stretch into the segment immediately downstream of it. This stretching dramatically increases the surface area between each segment of fluid. Due to the different species of fluids being miscible in each other, the increase in surface area increases the rate of diffusion and therefore the potential cross contamination between segments. As a result, downstream measurements that require significant sections of non-diffused fluids may become difficult or impossible to perform due to contamination.
Accordingly, a need exists in the art for systems and methods to ensure that samples can be obtained in microfluidic systems that are free or substantially free from contamination by diffused fluids.